Being first reported in 2001, store-operated Ca2+ entry (SOCE) is a relatively new phenomenon in skeletal muscle. SOCE is coordinated by coupling between two proteins: STIM1 calcium sensors in the sarcoplasmic reticulum (SR) and Ca2+-permeable Orai1 channels in the transverse tubule (TT) membrane. SOCE enhances muscle growth/development, limits fatigue, and promotes fatigue-resistant type I fiber specification. On the other hand, SOCE dysfunction contributes to muscle weakness/fatigue in aging, exacerbates muscular dystrophy, and mutations in STIM1 and Orai1 genes result in debilitating myopathies. The picture that emerges is that tight regulation of STIM1/Orai1-dependent SOCE activity is critical for optimal muscle performance such that increases or decreases in SOCE activity can lead to muscle fatigue, sarcopenia, and myopathy. Thus, Orai1-dependent SOCE represents a provocative potential therapeutic target for muscular dystrophy. We recently demonstrated that SOCE promotes skeletal muscle growth, limits muscle fatigue, and exacerbates the severity of muscular dystrophy in dystrophin- (mdx) and ?-sarcoglycan-deficient (sgcd-/-) mice. For this R21 application, we developed tamoxifen-inducible, muscle-specific Orai1 knockout mice in order to determine the specific role of Orai1-dependent Ca2+ entry in skeletal muscle in the dystrophic phenotypes observed in mdx and sgcd-/- mice, established mouse models of Duchene Muscular Dystrophy and Limb Girdle Muscular Dystrophy, respectively. We also established a collaboration with CalciMedica Inc. to evaluate the efficacy of systemic delivery of 4 potent new investigational SOCE channel inhibitors in mitigating the myopathic phenotypes of mdx and sgcd-/- mice. We will use these new research tools and collaborations, together with a comprehensive multi-disciplinary experimental approach, to evaluate the efficacy of inhibiting Orai1-dependent SOCE as a therapeutic intervention for muscular dystrophy. Based on our published and preliminary data, we hypothesize that partial inhibition of Orai1-dependent Ca2+ entry in skeletal muscle provides protection against myopathy in mouse models of muscular dystrophy without enhancing susceptibility to muscle fatigue. The validity of this central hypothesis will be rigorously tested in two Specific Aims. Aim 1 will use tamoxifen-inducible, muscle-specific Orai1 knockout mice to determine the impact of partial post-developmental, muscle-specific reduction of SOCE on muscular dystrophy. Aim 2 will determine the therapeutic efficacy of systemic administration of new generation Orai1 channel inhibitors obtained through a collaboration from CalciMedica Inc. to reduce the dystrophic phenotypes of mdx and sgcd-/- mice. The results of this project will provide needed preclinical evidence regarding the therapeutic potential of targeting Orai1-dependent Ca2+ entry as a treatment for muscular dystrophy.